The ultimate goal of cardiac tissue engineering is the regeneration of dysfunctional myocardium by using cells, biomaterial scaffolds, growth factors, and bioreactors. Progress in this field consistently faces two major barriers to success: (i) insufficient organization and mechanical function of tissue engineered cardiac grafts (TECG) and (ii) inadequate cardiogenesis (i.e. inadequate survival, alignment, and differentiation of cardiomyocytes (CM)). These shortcomings are due in part to sub-optimal properties of previous scaffolds and the empirical schemes typically used to create TECG. The hypothesis of this work is that a three dimensional (3D) scaffold with rationally designed structural and mechanical features can enhance the functional assembly of TECG. The project leverages recent work demonstrating the use of modeling and experimental studies to design scaffolds and TECG with cardiac-mimetic structural and mechanical properties, and the use of perfusion bioreactors to improve CM survival and TECG contractility. In Aim 1 we will start with an accordion-like honeycomb scaffold made of poly(glycerol sebacate) (PGS) that we recently demonstrated matches in-plane mechanical responses of native myocardium in the physiologic regime and guides orientation of cultured CM. We will use predictive modeling to determine if a particular scaffold pore layout in combination with a cell-laden hydrogel yields a biomimetic graft. If modeling predicts feasibility, then 250 [unreadable]m thick PGS scaffolds with open pore layouts will be made by laser microablation and used as scaffolds for heart cell culture. Resulting TECG will be assessed for CM orientation, differentiation, contractility, and mechanical properties, and these data will be used with further modeling to optimize scaffold design. Specifically, we will optimize TECG contractility by systematic studies of in-plane scaffold mechanical properties and CM differentiation by varying PGS curing conditions, pore layout, PGS surface topology, and characteristics of the cell-laden hydrogel. In Aim 2, we will scale-up to a fully 3D TECG by perfusion bioreactor culture of heart cells on a PGS scaffolds with rationally designed, fully 3D pore networks. These scaffolds will be produced by combining laser microablation and membrane lamination technologies, seeded by entrapping heart cells in hydrogel, and cultured in a perfusion bioreactor. The development and contractility of TECG will be quantified and optimized by systematic studies of bioreactor operating conditions, including flow regimen and hydrodynamic shear, that will be selected based on CM survival, differentiation, contractility, and the overall TECG structural, electrical and mechanical properties. The broad, long-term project objective is the rational design of tissue engineered cardiac grafts that can improve the success of myocardial repair procedures. The proposed TECG are expected to enhance myocardial regeneration by (i) providing biomimetic mechanical properties to help restore cardiac mechanical function and (ii) improving the scale and efficacy of cell delivery to promote graft survival and integration.